The serine proteases are the pivotal regulators of a variety of cellular processes critical for normal homeostasis. In the gut, host intestinal serine proteases not only control digestion, but their misregulated activities contribute to the pathogenesis of GI inflammatory, autoimmune, and neoplastic diseases. Recent evidence suggests that the intestinal paracellular pathway with its tight junctions (tj), plays a key role in modulating gut functions in healthy indiividuals. We recently found that the type II transmembrane serine protease, matriptase, is localized at intercellular apical junctional complexes in polarized intestinal epithelial cells and that loss of matriptase compromises barrier integrity. Depletion of matriptase by RNA interference enhances paracellular permeability in polarized Caco-2 monolayers cultured in vitro. In mice, matriptase depletion compromises epithelial barrier function, measured by transepithelial resistance of ex vivo epithelial tissue segments, and dramatically enhances susceptibility to experimental colitis induced by dextran sodium sulfate (DSS). Matriptase activates several inactive protease zymogens, including urokinase plasminogen activator (uPA) and prostasin, and also activates pro-hepatocyte growth factor (HGF), which serves as a ligand for c-met, a receptor tyrosine kinase involved in gastrointestinal repair. We expect that matriptase misregulation could contribute to barrier dysfunction underlying the pathogenesis of multiple microbial, autoimmune and inflammatory bowel diseases. The proposed studies will test the hypothesis that matriptase participates in the formation and maintenance of the GI mucosal barrier, and protects against exacerbated inflammatory and autoimmune responses. These hypotheses will be tested in the following specific aims: (1) to investigate junctional complex assembly underlying the paracellular permeability defect in matriptase hypomorph mice;(2) to investigate molecular pathways involved in the coupling of matriptase to tight junction assembly and the paracellular pathway and (3) to investigate the role of matriptase in the restoration of mucosal barrier function after DSS-induced acute injury. At the completion of these aims, we will have obtained molecular insight into the intestinal paracellular permeability defect caused by matriptase deficiency and will have a better understanding of the role of matriptase in maintaining intestinal epithelial barrier integrity after acute injury.